Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging

An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of V. serpens. Testing of several market samples of V. serpens using the SCAR marker revealed successful identification of the genuine samples of V. serpens. This study, therefore, provides a proficient in vitro propagation protocol of V. serpens using leaf explants and a SCAR marker for the authentic identification of V. serpens. This study will be helpful for conservation of authentic V. serpens.


Introduction
Medicinal plants have long been used as an important source of life saving drugs for human beings. India is a rich source of medicinal plants, but rapid population growth and human activities are now destroying their natural environment. Various medicinal plants are facing extinction and they require some urgent steps for their conservation. Viola serpens (Violaceae) is an important medicinal plant [1]. It has a high medicinal value in skin diseases, bleeding piles, throat cancer, asthma, constipation, fever, headache, and cough [2,3]. In addition, V. serpens is the main ingredient of Joshanda, used in cough and cold [4]. India, China, Java, Ceylon, Philippines, and Thailand are the geographical distribution BAP; indole-3-acetic acid, IAA; indole-3-butyric acid, IBA; and kinetin, KIN) were supplemented with the MS medium (Supplementary Table S1). After inoculation of the explant, observations were taken from day 25 up to day 60. No callusing of the explants was reported when the plant growth reguators (PGRs) were supplied without any combination (Table 1). Only a 10% frequency of callus formation appeared on the MS medium augmented with 2.0 mg/L BAP (Supplementary Table S1 (Table 1). Subsequent subculture in the same medium (MS + BAP + 2,4-D or MS + BAP + 2,4-D + KIN) resulted in increased growth of the callus. The callus appeared green ( Figure 1A). Compared to both combinations, the best results for callusing were detected on the MS medium supplemented with 2.0 mg/L BAP in combination with 0.13 mg/L 2,4-D. Eighty-eight percent callus formation from the leaf explants was observed with this combination after 45 days of inoculation, whereas only about 45% callus was obtained on the MS + BAP + 2,4-D + KIN after 60 days of inoculation (Table 1).
Plants 2020, 9, x FOR PEER REVIEW 3 of 14 supplemented with the MS medium (Supplementary Table S1). After inoculation of the explant, observations were taken from day 25 up to day 60. No callusing of the explants was reported when the plant growth reguators (PGRs) were supplied without any combination (Table 1). Only a 10% frequency of callus formation appeared on the MS medium augmented with 2.0 mg/L BAP (Supplementary Table S1 (Table 1). Subsequent subculture in the same medium (MS + BAP + 2,4-D or MS + BAP + 2,4-D + KIN) resulted in increased growth of the callus. The callus appeared green ( Figure 1A). Compared to both combinations, the best results for callusing were detected on the MS medium supplemented with 2.0 mg/L BAP in combination with 0.13 mg/L 2,4-D. Eighty-eight percent callus formation from the leaf explants was observed with this combination after 45 days of inoculation, whereas only about 45% callus was obtained on the MS + BAP + 2,4-D + KIN after 60 days of inoculation (Table 1).

Induction of Multiple Shoots
Subculturing of the callus, obtained from MS + BAP + 2,4-D media, was carried out on MS medium containing auxin and cytokinin in different combinations ( Table 2). Initiation of shoot buds were observed on the MS medium containing 0.5 mg/L NAA and 1.5-2.5 mg/L KIN. The MS medium containing 0.5 mg/L NAA and 2.25 mg/L KIN gave the optimum 70% shoot regeneration frequency. Subculturing of the shoot clumps on this medium resulted in an incerased formation of the shoot. Fifth subculture passages on this culture medium resulted in the highest regeneration frequency (86%) of shoots and the highest number of shoots (12), which was higher than the combination of NAA (0.50 mg/L) along with BAP (1.0-2.0 mg/L) (Table 2, Figure 1B). Although, different concentration of Plants 2020, 9, 246 5 of 14 MS + NAA (1.0, 1.5 and 2.0 mg/L) and KIN (3.0, 3.5 and 4.0 mg/L) were tried, no further enhancement of shoot regeneration frequency was observed (Data not shown). Thus, the combination of MS + NAA (0.5 mg/L) and KIN (2.25 mg/L) was considered to be the optimal growth regulator combination for maximum shoot regeneration in V. serpens (Table 2).

In Vitro Rooting as Influenced by Plant Growth Regulators
In vitro grown shoots (5.1 cm) were cu, and placed on the MS medium alone without any PGR or with different concentrations of PGRs (IAA and IBA). Root induction was not observed when the shoot was transferred on the MS (Full or 1/2) medium alone. The highest frequency of rooting was observed after 2-3 weeks of inoculation on the MS medium containing various concentrations of IBA. Among the different concentrations of IBA, the highest percentage of rooting response (81%) along with the mean number of roots (5.6) and root length (1.87 cm) was found on the MS medium containing 2 mg/L IBA (Table 3, Figure 1C). Shoots transferred on the MS medium containing various concentrations of IAA exhibited a lesser number of root formations than IBA (Table 3).

Acclimatization
Elongated shoots with well-defined roots were used for acclimatization. Individual plantlets were taken from the flasks. These plantlets, after washing with tap water, were shifted to a mixture of sand, decomposed coffee husk, and soil in a ratio of 1:2:1 in sterilized pots. They were kept for 15 days under high humidity, i.e., 70-80%, to avoid desiccation. Thereafter, the plantlets were placed under 80% shade net for one month. Therafter, these plants were transferred to the field ( Figure 1D).

Sequence Characterized Amplified Region (SCAR) Development
Accessions of V. serpens were procured from different sources for RAPD (Randomly Amplified Polymorphic DNA) analysis ( Table 4). Out of 20 RAPD primers (Supplementary Table S2  A bright and distinct RAPD amplicon (~1135 bp) that was specific to all the six accessions of V. serpens, as obtained with the OPAA-04 RAPD primer ( Figure 2C), was eluted and cloned in the pGEM-T easy vector (Promega, Madison, WI, USA). The size of the recombinant DNA (vector + RAPD amplicon) was~4335 bp. The recombinant DNA successfully transformed in Escherichia coli. The isolated recombinant DNA was resolved on 1.0% agarose gel. The size of the isolated plasmid was~4335 bp. Restriction digestion analysis with EcoR1 revealed a band of~1135 and 3000 bp, confirming the cloning of the RAPD amplicon ( Figure 3). A bright and distinct RAPD amplicon (~1135 bp) that was specific to all the six accessions of V. serpens, as obtained with the OPAA-04 RAPD primer ( Figure 2C), was eluted and cloned in the pGEM-T easy vector (Promega, Madison, WI, USA). The size of the recombinant DNA (vector + RAPD amplicon) was ~4335 bp. The recombinant DNA successfully transformed in Escherichia coli. The isolated recombinant DNA was resolved on 1.0% agarose gel. The size of the isolated plasmid was ~4335 bp. Restriction digestion analysis with EcoR1 revealed a band of ~1135 and 3000 bp, confirming the cloning of the RAPD amplicon ( Figure 3).     Sanger sequencing of the recombinant DNA was done using SP6 and T7 primers ( Figure 4). Forward (Vio F) and reverse (Vio R) SCAR primers were designed taking the sequence of the RAPD amplicon ( Table 5). The expected fragment size of the amplified DNA with these primers was 285 bp. When the genomic DNA of all the accessions of V. serpens was amplified with these primers, a single and bright 285 bp fragment of DNA was obtained ( Figure 5). It confirmed that the designed SCAR primers are highly specific and sensitive towards V. serpens.
Sanger sequencing of the recombinant DNA was done using SP6 and T7 primers ( Figure 4). Forward (Vio F) and reverse (Vio R) SCAR primers were designed taking the sequence of the RAPD amplicon ( Table 5). The expected fragment size of the amplified DNA with these primers was 285 bp. When the genomic DNA of all the accessions of V. serpens was amplified with these primers, a single and bright 285 bp fragment of DNA was obtained ( Figure 5). It confirmed that the designed SCAR primers are highly specific and sensitive towards V. serpens.           To test the applicability of the SCAR marker for the authentication of V. serpens, the SCAR primers were used to amplify genomic DNA of commercial crude drug market samples. The market samples in the lane numbers 1-6 of agarose gel showed a single and distinct DNA band of 285 bp ( Figure 6). No amplified product was seen in the lane numbers 7-9 of the agarose gel ( Figure 6). Therefore, it was found that the developed SCAR primers were able to amplify the genomic DNA of Viola serpens from the commercially available crude drug (Lanes 1-6). Absence of the amplified product in the rest of the lanes showed that the commercial crude drug did not contain Viola serpens. These results show the commercial applicability of the SCAR primers and, thus, it can be used for the molecular authentication of medicinal plants.
To test the applicability of the SCAR marker for the authentication of V. serpens, the SCAR primers were used to amplify genomic DNA of commercial crude drug market samples. The market samples in the lane numbers 1-6 of agarose gel showed a single and distinct DNA band of 285 bp ( Figure 6). No amplified product was seen in the lane numbers 7-9 of the agarose gel ( Figure 6). Therefore, it was found that the developed SCAR primers were able to amplify the genomic DNA of Viola serpens from the commercially available crude drug (Lanes 1-6). Absence of the amplified product in the rest of the lanes showed that the commercial crude drug did not contain Viola serpens. These results show the commercial applicability of the SCAR primers and, thus, it can be used for the molecular authentication of medicinal plants.

Discussion
Increases in the population and rapid technological advances are putting tremendous pressure on natural genetic resources causing wild resources of medicinal plants to diminish rapidly. Micropropagation is a key tool of plant biotechnology that exploits the totipotent nature of plant cells, a concept proposed by Haberlandt [14]. In vitro micropropagation of valuable plant species plays a major role in the conservation of the germplasm, rapid clonal propagation of genetically-manipulated elite clones, production of secondary metabolites, establishment of extensive collections using minimum space, and the supply of valuable material for wild population recovery [10,15]. This technique is widely used for the commercial propagation of various medicinal plants [16,17]. In the present study, in vitro micropropagation protocol of V. serpens was established using leaf explants because this plant is widely used in a large number of herbal formulations and the procurement of this plant is carried out mainly from wild resources. Earlier, plant propagation protocols for V. patrinii [18,19] and V. ulginosa [20] were established. In vitro propagation of V. serpens was also carried out earlier using petiole explants [4]. In our study, leaf explants were inoculated on MS medium without and with varying concentrations of PGRs (NAA, KIN, BAP, 2,4-D, IAA) singly. No positive response in the callusing of the explants was found. The addition of auxin along with cytokinin produced positive results in V. serpens. Among the different combination tested, supplementation with a combination of BAP (2.0 mg/L) and 2,4-D (0.13 mg/L) with the MS medium resulted in the best callus induction, showing about 90% callus formation from the leaf explants. Further, the MS medium augmented with 0.5 mg/L NAA and 2.25 mg/L KIN gave the best results in our study for shoot proliferation from the callus. In contrast with our results, Soni and Kaur [21] showed that MS medium in combination with BA (1.0 mg/L) and KIN (0.25 mg/L) exhibited the best response for in vitro shoot regeneration in V. pilosa. Vishwakarma et al. [4] reported that the petiole explants did not show any ~ 285 bp

Discussion
Increases in the population and rapid technological advances are putting tremendous pressure on natural genetic resources causing wild resources of medicinal plants to diminish rapidly. Micropropagation is a key tool of plant biotechnology that exploits the totipotent nature of plant cells, a concept proposed by Haberlandt [14]. In vitro micropropagation of valuable plant species plays a major role in the conservation of the germplasm, rapid clonal propagation of genetically-manipulated elite clones, production of secondary metabolites, establishment of extensive collections using minimum space, and the supply of valuable material for wild population recovery [10,15]. This technique is widely used for the commercial propagation of various medicinal plants [16,17]. In the present study, in vitro micropropagation protocol of V. serpens was established using leaf explants because this plant is widely used in a large number of herbal formulations and the procurement of this plant is carried out mainly from wild resources. Earlier, plant propagation protocols for V. patrinii [18,19] and V. ulginosa [20] were established. In vitro propagation of V. serpens was also carried out earlier using petiole explants [4]. In our study, leaf explants were inoculated on MS medium without and with varying concentrations of PGRs (NAA, KIN, BAP, 2,4-D, IAA) singly. No positive response in the callusing of the explants was found. The addition of auxin along with cytokinin produced positive results in V. serpens. Among the different combination tested, supplementation with a combination of BAP (2.0 mg/L) and 2,4-D (0.13 mg/L) with the MS medium resulted in the best callus induction, showing about 90% callus formation from the leaf explants. Further, the MS medium augmented with 0.5 mg/L NAA and 2.25 mg/L KIN gave the best results in our study for shoot proliferation from the callus. In contrast with our results, Soni and Kaur [21] showed that MS medium in combination with BA (1.0 mg/L) and KIN (0.25 mg/L) exhibited the best response for in vitro shoot regeneration in V. pilosa. Vishwakarma et al. [4] reported that the petiole explants did not show any sign of callus formation on MS medium supplemented with different PGRs (NAA, BAP, KIN, IAA) singly in V. serpens. However, the addition of auxin along with cytokinin gave positive results in petiole explant. The MS medium supplemented with 2,4-D produced a significant result using unfertilized ovules in Viola odorata [22]. Chalgeri et al. [18] reported no response on callus induction of V. patrinii with higher concentrations of NAA and BAP. In addition, transfer of regenerated shoot on half-strength MS medium augmented with IBA induced root formation, where 73% rooting with a 3.6 average number of roots and 1.3 cm root length was observed. These results are better than earlier reports on root induction in V. serpens using IAA [4]. In Viola patrinii, development of significant roots were obtained in 2-fold diluted MS media supplemented with 9.85 µM IBA and 2% (w/v) sucrose [18]. The highest rooting frequency was obtained on MS medium supplemented with 2.0 mg/L IBA in our study after 2-3 weeks of transfer. Complete plantlets were successfully acclimatized in the culture room and then transferred in field conditions. The in vitro-micropropagated plants successfully survived the natural environmental conditions in the field, indicating the success of this protocol for plant regeneration of V. serpens using leaf explants.
In addition to the development of an efficient in vitro propagation protocol for V. serpens, a DNA-based technique for the authentication of genuine V. serpens was also developed in this study in view of the need for a tool that can distinguish a genuine plant from the adulterated samples. The differences that distinguish one species from another are the deoxyribonucleic acids (DNA), which are encoded in the genetic material of the species and these DNA-based methods are one of the most reliable methods for the authentication of plants. Thus, these DNA-based markers are unique and stable as the DNA of every individual is unique in nature. Many molecular markers, viz., RAPD, RFLP (Restriction Fragment Length Polymorphism), and AFLP (Amplified Fragment Length Polymorphism), have been used for the DNA analysis of medicinal plants. These molecular markers are easy to develop and are simple markers but the lack of reproducibility makes them less reliable for the identification of the genuine plant. To overcome these drawbacks, a more specific marker (i.e., SCAR marker) was developed as it was single locus-specific and its PCR amplification was not very sensitive to the reaction conditions. Conversion of molecular markers, like RAPD and RFLP, to the sequenced data-based SCAR marker increases the reproducibility of PCR products. Thus, this marker is a fast and more reliable method for the authentication of the plant. In the present study, a SCAR marker was developed for the identification of V. serpens. SCAR markers have also been used to study adulteration in medicinal plants like Cynanchum [23], Echinacea [24], Pueraria [25], Gardenia jasminoides [26], and Phyllanthus species [27]. Authentication of commercially important food and spice products, like ground chili [28], has also been done using SCAR markers. Viola serpens is a priority-listed medicinal plant. Because of over exploitation, availability of this plant is continuously decreasing in the wild. Thus, development of a SCAR marker for its identification will help to conserve natural resources. Authentication of endangered plants, like Michelia coriacea [29], Commiphora spp. [30], and Physalis [31], has also been carried out earlier using SCAR markers.

Collection of Plant Materials
For micropropagation, authentic plants of Viola serpens were procured from Jogindernagar, Himachal Pradesh, India and grown at Jamia Hamdard herbal garden, New Delhi. For developing the SCAR marker, plants were collected from six different locations (Table 4). Authentication was done by the Botanical Survey of India, Dehradun (India) with a voucher herbarium specimen No. 114835 of V. serpens.
The callus obtained was subcultured on the MS medium supplemented with different concentrations of NAA, KIN, BAP alone, and a combination of NAA + BAP or NAA + KIN for shoot differentiation. Emerging shoots from explants were inoculated on half-or full-strength MS medium supplemented with varying concentrations of IAA and IBA (0.5, 1.0, 1.5, 2.0, and 2.5 mg/L) for rooting. Maintenance of the cultures was carried out at 25 • C ± 2 • C with an 8/16 h (light/dark) photoperiod.
Individual plantlets of V. serpens were excised from the flasks, then washed with tap water, and transferred to a mixture of sand, decomposed coffee husk, and soil in a ratio of 1:2:1 in a sterilized pot. High humidity (80-90%) conditions were kept for 15 days to prevent desiccation. The experiments were carried out, taking six replicates, with each plant growth regulator. Data from the experiment were analyzed by calculating the mean and standard error using SYSTAT 13 software (Stat Soft Inc., Tulsa, OK, USA).

Development of Sequence Characterized Amplified Region (SCAR)
Genomic DNA isolation of Viola serpens was carried out by Doyle and Doyle [33]. Twenty random primers (15 from Operon technologies, Qiagen, Germantown, MD, USA and five primers from BangloreGenei, Bengaluru, India) were initially screened for RAPD analysis (Supplementary Table S2). Based on the ability to detect distinct polymorphic amplified products across the accessions, primers were selected for further analysis. The primers that generated weak products were discarded to ensure reproducibility. The RAPD reaction was performed by the method developed by McClelland et al. [34]. The polymerase chain reactions consisted of reaction buffer (20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.5 U of Taq polymerase), 300 mM dNTPs (deoxyribonucleotides), 1.5 mM MgCl 2 , 25 pM primer, and 25 ng template DNA. Amplification conditions were set as an initial denaturation temperature of 95 • C for 4 min, followed by 40 cycles of 94 • C (30 s), 34 • C (50 s), and 72 • C (60 s) in a thermal cycler (Master cycler, Eppendorf, USA). Amplified products were run on 1.0% agarose gel in 1×TAE buffer. A standard 100 bp DNA ladder was also run along with the amplified products. Staining was done by 0.5 µg/mL ethidium bromide. Visualization and photography of the resolved DNA were carried out using a gel documentation system (Alpha innotech, San Leandro, CA, USA). A 1135 bp-sized DNA fragment of V. serpens was excised from the agarose gel and eluted using a gel extraction kit (Qiagen, Germantown, MD, USA). Cloning and sequencing of the eluted amplicon were further performed. A-tailing to the amplicon was carried out using the eluted DNA fragment, reaction buffer (1.5 mM MgCl 2 , 0.2 mM dATP), and 1 unit of Taq polymerase. The reaction was incubated for 30 min at 70 • C. These A-tailed DNA were ligated into the pGEM-T easy vector (Promega, Madison, WI, USA) following the manufacturer's instruction. The recombinant DNA was transformed into E. coli DH10β. Few distinct colonies were picked and grown on Lauri-Bertani medium containing ampicillin until the OD 600 reached 0.3. The recombinant DNA was isolated using a plasmid isolation kit (Qiagen, Hilden, Germany). Sequencing of the cloned DNA fragment was performed with SP6 and T7 primers at the Centre for Genomic Application, New Delhi, India. Forward primer (Vio F) and reverse primer (Vio R) for developing the SCAR marker were designed (Table 5). GC contents, secondary structures, and the melting temperature of each primer were also determined. These sequences were custom synthesized by IDT Technologies, USA. The in-house designed SCAR primers were used for the amplification of the genomic DNA of genuine plant materials. Nine samples from a local market in New Delhi, India were procured, which were sold under the name Banafsa (local name of V. serpens) (Supplementary Table S4). Genomic DNA of these samples was isolated and amplification of the isolated DNA was carried out using SCAR primers (Vio F and Vio R) under similar PCR conditions. The amplified DNA products were run on 1.0% agarose gel. A standard 100 bp DNA ladder was also run along with the amplified DNA products. Staining was carried out using 0.5 µg/mL ethidium bromide. Visualization and photography of the resolved DNA were carried out using a gel documentation system (Alpha innotech, San Leandro, CA, USA).

Conclusions
The current investigation provided a promising technique for an efficient regeneration protocol of V. serpens using leaf explants. The PGR-free MS medium and the MS medium combined with varying concentrations of single plant growth regulators (NAA, BAP, KIN, IAA, 2,4-D) showed negligible callusing on the explants. The MS medium along with BAP (2.0 mg/L) in combination with 2,4-D (0.13 mg/L) was the best for callusing, where about 90% callus were induced from the leaf explants. Further, the MS medium augmented with NAA (0.5 mg/L) and KIN (2.25 mg/L) gave the best results for shoot proliferation from the callus. Regenerated shoots were rooted on various concentration of IBA. Among the different concentrations of IBA tested, the highest rooting frequency was obtained on MS medium supplemented with IBA (2.0 mg/L) after 2-3 weeks of transfer. Complete plantlets were successfully acclimatized in the culture room and then transferred to the field. Further, a reliable and reproducible marker (SCAR marker) was developed for the identification of genuine samples of the V. serpens. This marker can specifically identify the genetic materials of V. serpens from other plant species. Therefore, the development of an efficient regeneration protocol and the SCAR marker of V. serpens will greatly contribute to the ecological conservation of this important medicinal plant.
Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/2/246/s1, Table S1: Effect of different plant growth regulators (PGRs) on leaf explants of Viola serpens for callus formation, Table S2: RAPD primers along with sequences, Table S3: Number of amplification products generated by 20 arbitrary primers in different accessions of Viola serpens, and Table S4: Commercial crude drug samples collected from the market place of New Delhi, India.